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- EMDB-8633: CryoEM Structure of a Prokaryotic Cyclic Nucleotide-Gated Ion Channel -

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Basic information

Entry
Database: EMDB / ID: EMD-8633
TitleCryoEM Structure of a Prokaryotic Cyclic Nucleotide-Gated Ion Channel
Map dataUnsharpened map
Sample
  • Complex: LliK
    • Protein or peptide: LliK
Function / homology
Function and homology information


HCN channel complex / regulation of membrane depolarization / voltage-gated potassium channel activity / sodium ion transmembrane transport
Similarity search - Function
: / Potassium channel, voltage-dependent, EAG/ELK/ERG / Cyclic nucleotide-binding domain signature 2. / Cyclic nucleotide-binding, conserved site / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily / RmlC-like jelly roll fold ...: / Potassium channel, voltage-dependent, EAG/ELK/ERG / Cyclic nucleotide-binding domain signature 2. / Cyclic nucleotide-binding, conserved site / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily / RmlC-like jelly roll fold / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
Transporter, cation channel family / cyclic nucleotide-binding domain multi-domain protein
Similarity search - Component
Biological speciesLeptospira licerasiae serovar Varillal str. VAR 010 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsJames ZM / Borst AJ / Haitin Y / Frenz B / DiMaio F / Zagotta WN / Veesler D
CitationJournal: Proc Natl Acad Sci U S A / Year: 2017
Title: CryoEM structure of a prokaryotic cyclic nucleotide-gated ion channel.
Authors: Zachary M James / Andrew J Borst / Yoni Haitin / Brandon Frenz / Frank DiMaio / William N Zagotta / David Veesler /
Abstract: Cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-regulated (HCN) ion channels play crucial physiological roles in phototransduction, olfaction, and cardiac pace making. ...Cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-regulated (HCN) ion channels play crucial physiological roles in phototransduction, olfaction, and cardiac pace making. These channels are characterized by the presence of a carboxyl-terminal cyclic nucleotide-binding domain (CNBD) that connects to the channel pore via a C-linker domain. Although cyclic nucleotide binding has been shown to promote CNG and HCN channel opening, the precise mechanism underlying gating remains poorly understood. Here we used cryoEM to determine the structure of the intact LliK CNG channel isolated from -which shares sequence similarity to eukaryotic CNG and HCN channels-in the presence of a saturating concentration of cAMP. A short S4-S5 linker connects nearby voltage-sensing and pore domains to produce a non-domain-swapped transmembrane architecture, which appears to be a hallmark of this channel family. We also observe major conformational changes of the LliK C-linkers and CNBDs relative to the crystal structures of isolated C-linker/CNBD fragments and the cryoEM structures of related CNG, HCN, and KCNH channels. The conformation of our LliK structure may represent a functional state of this channel family not captured in previous studies.
History
DepositionMar 10, 2017-
Header (metadata) releaseMar 22, 2017-
Map releaseApr 12, 2017-
UpdateJul 18, 2018-
Current statusJul 18, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.024
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.024
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5v4s
  • Surface level: 0.024
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8633.png

Downloads & links

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Map

FileDownload / File: emd_8633.map.gz / Format: CCP4 / Size: 47.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.37 Å/pix.
x 232 pix.
= 316.796 Å		1.37 Å/pix.
x 232 pix.
= 316.796 Å		1.37 Å/pix.
x 232 pix.
= 316.796 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.3655 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.024 / Movie #1: 0.024
Minimum - Maximum-0.023005761 - 0.061287668
Average (Standard dev.)0.0003627006 (±0.0031903565)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions232232232
Spacing232232232
CellA=B=C: 316.796 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.36551.36551.3655
M x/y/z232232232
origin x/y/z0.0000.0000.000
length x/y/z316.796316.796316.796
α/β/γ90.00090.00090.000
start NX/NY/NZ-152-37
NX/NY/NZ998271
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS232232232
D min/max/mean-0.0230.0610.000

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Supplemental data

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Sample components

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Entire : LliK

EntireName: LliK
Components
  • Complex: LliK
    • Protein or peptide: LliK

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Supramolecule #1: LliK

SupramoleculeName: LliK / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Leptospira licerasiae serovar Varillal str. VAR 010 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 200 KDa

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Macromolecule #1: LliK

MacromoleculeName: LliK / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Leptospira licerasiae serovar Varillal str. VAR 010 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKHHHHHHHH PMSDVDIPTT ENLYFQGSGS MGITLKNRIR VYWDILVFIC IFWASLESPL RIVINYDPNL LLTCIYFFID FVFALDIL W NCFTPEYKDG KWILTRSQVI KDYLGSWFII DLIAALPLEY ATTTIFGLQQ SQYPYLYLLL GVTRILKVFR ISDILQRINL ...String:
MKHHHHHHHH PMSDVDIPTT ENLYFQGSGS MGITLKNRIR VYWDILVFIC IFWASLESPL RIVINYDPNL LLTCIYFFID FVFALDIL W NCFTPEYKDG KWILTRSQVI KDYLGSWFII DLIAALPLEY ATTTIFGLQQ SQYPYLYLLL GVTRILKVFR ISDILQRINL AFQPTPGI L RLVLFAFWAT LVAHWCAVGW LYVDDLLDYQ TGWSEYIIAL YWTVATIATV GYGDITPSTD SQRIYTIFVM ILGAGVYATV IGNIASIL G SLDLAKAAQR KKMAQVDSFL KARNISQNIR RRVRDYYMYI IDRGWGEDEN ALLNDLPISL RREVKIQLHR DLLEKVPFLK GADPALVT S LVFSMKPMIF LEGDTIFRRG EKGDDLYILS EGSVDILDSD EKTILLSLQE GQFFGELALV MDAPRSATVR ATTTCEIYTL SKTDFDNVLK RFSQFRSAIE ESVAHLKKR

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.9 mg/mL
BufferpH: 7.9
Component:
ConcentrationNameFormula
20.0 mMTris
150.0 mMPotassium ChlorideKCl
0.05 mMLMNG
0.005 mMCholesterol Hemi-Succinate
0.02 mMcAMP
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 1.4 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 379000
CTF correctionSoftware - Name: Gctf
Startup modelType of model: OTHER / Details: e2initialmodel.py
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 18737
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.0)
Final 3D classificationSoftware - Name: RELION (ver. 2.0)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-5v4s:
CryoEM Structure of a Prokaryotic Cyclic Nucleotide-Gated Ion Channel

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